NEW USE OF BCG IMMUNOGENIC FORMULATION EXPRESSING A RESPIRATORY SYNCITIAL VIRUS PROTEIN AGAINST hMPV

ABSTRACT

The invention relates to the novel use of an immunogenic formulation containing the  bacillus  Calmette-Guérin (BCG) strain at a concentration between 104-109 bacteria, expressing at least one protein or immunogenic fragment of respiratory syncytial virus (RSV, Human orthopneumovirus), in a pharmaceutically acceptable saline buffer solution because it serves to prepare a vaccine useful to prevent, treat, or attenuate human metapneumovirus (hMPV) infections.

FIELD OF THE INVENTION

An immunogenic formulation useful for preparing a vaccine against respiratory viruses, such as respiratory syncytial virus (RSV, Human orthopneumovirus) and/or human metapneumovirus (hMPV), is disclosed, where this formulation comprises at least one attenuated strain of Mycobacterium, preferably a strain Bacillus Calmette-Guérin (BCG), which recombinantly expresses one or more proteins or immunogenic fragments of RSV.

BACKGROUND OF THE INVENTION

Among the respiratory viruses, respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) stand out for their clinical importance, as they can cause very severe infections in infants and young children. The clinical manifestations of both viruses are indistinguishable from each other, causing a wide range of clinical cases, which can be mild, such as rhinitis, or much more severe, such as pneumonia or bronchiolitis, with the most serious conditions being observed in infants, premature infants, children with congenital heart disease. and in immunosuppressed patients. RSV is the main causative agent of acute respiratory tract infections in infants worldwide. According to the WHO, this virus infects 64 million people annually, of which 160,000 die. RSV is the leading cause of infection and hospitalization in children under 2 years of age. (www.who.int). For its part, human metapneumovirus is the second leading cause of hospitalizations due to acute respiratory infections in children under 5 years of age.

Infections caused by these viruses are extremely frequent and recurrent since practically 100% of children older than three years have presented at least one episode of RSV and/or hMPV infection at 5 years of age. Because these infections do not leave a complete immune memory, reinfections are frequent, decreasing in severity as the age of the patient increases.

The health situation generated by these respiratory infections has a high economic impact for the affected countries. Studies carried out in developed countries estimate that the individual cost of RSV infection is over 3,000 euros (approximately $2,250,000 Chilean pesos), with an upper limit of up to 8,400 euros (approximately $6,800,000 Chilean pesos).

Both RSV and hMPV are classified within the Pneumoviridae Family, a family that has two genera, Metapneumovirus and Orthopneumovirus. While hMPV belongs to the genus Metapneumovirus, RSV belongs to the genus Orthopneumovirus.

Both viruses contain non-segmented negative single-stranded RNA, with lipid envelope and similar, but not identical, organization. RSV has a genome of approximately 15 kb, with 10 genes that code for a total of 11 proteins. Five of these proteins have structural functions, corresponding to the transmembrane glycoproteins F, G, and SH, the nucleocapsid N protein, and the matrix M protein. The other four proteins, M2-1, M2-2, P, and L are involved in viral replication and transcription. The two remaining proteins, designated NS1 and NS2, are non-structural and are involved in virulence. On the other hand, hMPV has a genome of approximately 13 kb and comprises 8 genes that code for nine proteins: N, P, M1, F, M2-1, M2-2, SH, G, and L. hMPV does not contain the NS1 and NS2 proteins. The N protein is the most conserved among both viruses, RSV and hMPV.

RSV that infects humans has different strains or subtypes, with subgroups A and B being those that predominate in the population. The main antigenic difference between the subgroups corresponds to the G protein, which only conserves 40-44% of the amino acids between the different subgroups.

Due to the differences that exist between the two viruses, it is not obvious or predictable that a single vaccine could provide simultaneous protection for RSV and hMPV. As we will explain in detail later, the vaccine of the invention is a formulation containing the Bacillus Calmette-Guérin (BCG) strain, in a concentration of between 1×10⁴ a 1×10⁹ colony-forming units per dose, which expresses at least one protein or immunogenic fragment of respiratory syncytial virus (RSV). Surprisingly, the inventors have found that this immunogenic formulation confers protection against both RSV and hMPV infections.

In the state of the art, we find some unsuccessful attempts to procure a vaccine against RSV. An attempt made in the 1960s consisted of formalin-inactivated whole virus (RSV-FI), which was administered intramuscularly to volunteers in the presence of adjuvant alum (1). Contrary to expectations, this immunization produced a much more severe respiratory condition in vaccinated children after natural RSV infection, which led to the hospitalization of 80% of those vaccinated and the death of two of them (2). The respiratory-pulmonary picture presented by the vaccinated was characterized by an unusual infiltration of eosinophils and neutrophils, together with a high titer of complement-fixing antibodies (2). Analysis of the lung tissues of children vaccinated with RSV-FI who died due to RSV infection shows the deposition of complement, immune complexes, and the presence of eosinophils in the peribronchial regions (3). Along with this, studies in animal models show that vaccination with RSV-FI produces a Th2-type immune response, based on CD4+ T lymphocytes. These same characteristics have been observed in animals that have been immunized with the G protein (4) or that have received CD4+ T lymphocytes specific for this glycoprotein prior to infection with RSV.

In this same sense, the Th2 response is not capable of conferring protection against sustained viral infection over time, and stimulates, through the secretion of IL-5, the eosinophils responsible for allergic reactions and inflammatory hyper-reactivity, which can even be more dangerous than the viral infection. In this type of Th2 response, T cells also secrete IL-4, a cytokine that induces the generation of IgG1 and IgE isotype antibodies, inhibiting the IgM, IgG3, IgG2a, and IgG2b isotypes.

For these reasons, an effective and safe vaccine against RSV and hMPV respiratory viruses should generate a Th1-type immune response, based on CD4+ T helper lymphocytes and interferon-gamma (IFN-γ) secreting cytotoxic T cells. This response is capable of conferring sustained protection overtime against these viral infections, also inducing the proliferation and differentiation of B cells secreting antibodies of the IgG2a isotype and inhibiting the production of immunoglobulins of the IgG3, IgE, IgG2b, and IgG1 isotypes.

Ongoing research on RSV vaccines has focused on the analysis and development of viral subunits, such as the F proteins (5), M2 (6), and also some conserved segments of the G protein (7). On the other hand, the generation of vaccines based on mutant strains of RSV, such as those sensitive to temperature (8), with deletion of some genes (9) or recombinants for cytokines such as GM-CSF (10) have also been studied. Some of these vaccines have been tested in phase I and II clinical trials, with variable results (11-13). Another type of tentative vaccine against RSV is the case of vaccines based on proteins F and G, which are administered with adjuvants such as ISCOMs. Immunization with this type of vaccine produces an increased infiltration of eosinophils in the lung tissue against a new viral infection (14), which increases the damage in the lung tissue.

The infant's immune system is characterized by preferentially developing Th2-type immune responses, possibly due to the immaturity of the immune system during the first six months of life (15, 16). However, if properly stimulated, it can mount a Th1-type response (17).

In order to formulate an effective and safe vaccine against RSV and/or hMPV, the inventors have carried out a finished study of the immune response generated against the different RSV proteins, in order to identify those that allow the generation of a Th1-type immune response, based on cytotoxic T lymphocytes. The use of bacterial vectors for the heterologous expression of viral antigens has the advantage that they can be used as live attenuated vectors since they have their invasion capacities intact and are recognized as non-pathogenic. An additional advantage of certain bacterial vectors used for the expression of heterologous antigens is their recognized ability to induce Th1-type immunities (18, 19), which is highly attractive in the case of vaccine development. Bacillus Calmette-Guérin (BCG) is an attenuated strain of Mycobacterium bovis that is used as a vaccine in newborns against Mycobacterium tuberculosis. Since BCG was approved as a tuberculosis vaccine, it has been given to more than 3.3 billion people around the world. Its massive use has been facilitated by several advantageous characteristics of this bacterium, such as its great thermostability in the lyophilized form. Furthermore, immunizing newborn infants with this bacterium is not risky and produces only minimal side effects. Regarding the safety and stability of BCG reversion to virulence has never been observed over all the years it has been in use. BCG is highly immunogenic and with just one dose it is possible to generate an immune response that is maintained for long periods. Importantly, BCG induces a potent Th1-type immune response in both adults and children (20). This phenomenon in neonates is evidenced by the cellular-type immune response that is generated against M. tuberculosis antigens (PPD), a response that manages to be maintained for prolonged periods (20).

To date, several bacterial, parasitic, and viral antigens have been successfully expressed in this bacterial system, which when evaluated in animal models have been shown to be capable of generating humoral and cellular immunity against these antigens (21, 22). In addition, BCG has the particularity of not being neutralized by antibodies present in breast milk, so it can be used as an immunity inducer in nursing children (22). The present invention corresponds to an immunological formulation that contains one or more attenuated recombinant strains of Mycobacterium, bacteria, preferably the BCG strain, for RSV proteins and can be used in the preparation of vaccines against respiratory viruses, especially RSV and hMPV.

The formulation of the invention makes it possible to avoid or attenuate the lung damage caused by infection by the respiratory viruses RSV and hMPV, thanks to the generation of an efficient and favorable immune response for the elimination of the virus. Because attenuated strains of Mycobacterium, such as BCG, are potent inducers of Th1-type immune responses, the immune response induced by recombinant BCG strains for RSV provides protection against infections caused both viruses.

BRIEF DESCRIPTION OF THE INVENTION

The present invention consists of an immunogenic formulation that induces protection against infection caused by respiratory viruses, especially infections caused by respiratory syncytial virus (RSV) and/or human metapneumovirus (hMPV), or at least attenuates the pathologies caused by these viruses in mammals. The immunogenic formulation of the present invention can be used to prepare vaccines and contains colony forming units (CFU) of live attenuated recombinant strains of Mycobacterium, preferably the Bacillus Calmette-Guérin (BCG) strain that recombinantly or heterologously express one or more proteins or immunogenic fragments of respiratory syncytial virus.

These proteins or immunogenic fragments of RSV correspond to the RSV NS1, NS2, N, P, M, SH, M2 (ORF1), M2 (ORF2), L, F or G proteins of the RSV A or RSV B subtypes and are found inserted either in the bacterial genome of the attenuated strains of Mycobacterium or in extrachromosomal plasmids, in one or more copies, and their expression is managed by endogenous or exogenous, constitutive or inducible, BCG promoters. These proteins or immunogenic fragments of RSV can be expressed by BCG or other attenuated strains of Mycobacterium, in soluble-cytoplasmic form, secreted extracellularly or as proteins bound to the cell membrane.

The immunogenic formulation disclosed in the present invention can be used in conjunction with immunogenic formulations that contain other attenuated strains of Mycobacterium or BCG and that differ in the immunogenic RSV proteins that they express, as well as in the location of the genes (inserted in the genome or extrachromosomal), the number of copies of the gene protein, the promoter that induces the expression of the protein, or the destination of the RSV immunogenic protein or fragments (soluble-cytoplasmic, extracellularly secreted or cell membrane-bound proteins).

The immunogenic formulation described above is useful for preparing a vaccine, which can be applied to the individual subcutaneously, percutaneously, or subdermally, in conjunction with a buffered or physiological saline solution.

The immunogenic formulation of the invention can be used to vaccinate individuals who have or have not had previous contact with a respiratory syncytial virus or human metapneumovirus, to confer protection against respiratory viruses, such as RSV and/or hMPV.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the percentage of CD8+/CD69+ cells (A) and (B) IFN-7 secretion of cells from the spleen of BALB/c mice immunized with BCG in saline solution ((PBS: 137 mM NaCl; 2.7 mM KCl; 4.3 mM Na2HPO4; 1.47 mM KH2PO4, Tween 80 0.02% pH 7.4, a 4° C.) that expresses the N protein. 5×10⁵ cells from the spleen of unimmunized animals, immunized with 1×10⁷ PFU of RSV, immunized with BCG (1×10⁸ CFU/mouse in PBS: 137 mM NaCl; 2.7 mM KCl; 4.3 mM Na₂HPO₄; 1.47 mM KH₂PO₄, Tween 80 0.02% pH 7.4, 4° C.) or immunized with recombinant BCG for the RSV N protein (1×10⁸ CFU/mouse in PBS: 137 mM NaCl; 2.7 mM KCl; 4.3 mM Na₂HPO₄; 1.47 mM KH₂PO₄, Tween 80 0.02% pH 7.4, 4° C.) stimulated for 72 hrs. with 0.5 μM of RSV N protein. Subsequently, the percentage of positive cells for the CD8 and CD69 (A) markers was determined by flow cytometry. Cell supernatants were subjected to ELISA to detect the presence of secreted IFN-γ. (B) **, p value 0.002, Student's t-test. It can be concluded that the recombinant BCG strain for the RSV N protein produces, in mice immunized with this immunogenic formulation, a favorable response by T lymphocytes.

These results show that in response to immunization with recombinant BCG for RSV N protein, these CD8+ T lymphocytes are activated, since they express activation markers on their surface (CD69+) and secrete IFN-γ to the extracellular medium, which evidence that the vaccine of the invention generates a Th1-type immune response.

FIG. 2 shows a curve of body weight variation of BALB/c mice immunized with a recombinant BCG strain that express RSV N protein (1×10⁸ CFU/mouse, in PBS: 137 mM NaCl; 2.7 mM KCl; 4.3 mM Na₂HPO₄; 1.47 mM KH₂PO₄, Tween 80 0.02% pH 7.4, 4° C.) or with a recombinant BCG strain for RSV M2 protein (1×10⁸ CFU/mouse, in PBS: 137 mM NaCl; 2.7 mM KCl; 4.3 mM Na₂HPO₄; 1.47 mM KH₂PO₄, Tween 80 0.02% pH 7.4, 4° C.). Unvaccinated BALB/c mice (▪), immunized with 1×10⁷ plaque-forming units (PFU) of UV-inactivated RSV (UV Bulb, 312 nm, power 8 watts) for 20 minutes (RSV-UV) (▴), immunized with a strain of wild BCG (WT), which does not express RSV (▾) (1×10 CFU/mouse, in PBS: 137 mM NaCl; 2.7 mM KCl; 4.3 mM Na₂HPO₄; 1.47 mM KH₂PO₄, Tween 80 0.02% pH 7.4, 4° C.), immunized with a BCG strain transformed with pMV361-N (□) (1×10⁸ CFU/mouse, in PBS: 137 mM NaCl; 2.7 mM KCl; 4.3 mM Na₂HPO₄; 1.47 mM KH₂PO₄, Tween 80 0.02% pH 7.4, 4° C.) or immunized with a BCG strain transformed with pMV361-M2 (Δ) (1×10⁸ CFU/mouse, in PBS: 137 mM NaCl; 2.7 mM KCl; 4.3 mM Na₂HPO₄; 1.47 mM KH₂PO₄, Tween 80 0.02% pH 7.4, 4° C.) were infected intranasally with 1×10⁷ PFU of RSV, strain 13018-8. As a control, a group of unvaccinated and uninfected mice (●) was included. The variation in body weight with respect to day 0 was recorded daily for 4 days. **, p-value 0.002, Student's t-test.

It can be concluded that the immunization of mice with a recombinant BCG strain for RSV N or M2 proteins produces a favorable response against RSV infection since post-infection the body weight of these mice does not vary significantly when compared to that of unvaccinated mice, in which a decrease in body weight is observed post-infection.

FIG. 3 -A shows the percentage of CD11c⁻/CD11b⁺/Gr1⁺ cells in bronchoalveolar lavage of BALB/c mice from four experimental groups: unimmunized and uninfected, unimmunized and intranasally infected with 1×10⁷ of hMPV strain cz0107, mice immunized with BCG recombinant for the RSV N protein (1×10⁸ CFU/mouse) and mice immunized with recombinant BCG for hMPV P protein (1×10⁸ CFU/mouse).

It is observed that the groups immunized with the recombinant BCG strain for the P protein of hMPV and the N protein of RSV show significantly less polymorphonuclear infiltration than the infected group without immunization.

FIG. 3 -B shows the percentage of CD11c⁻/CD11b⁺/Gr1⁺ cells in the lung for the four groups studied.

As in the previous case, it is observed that the groups immunized with the recombinant BCG strain for the P protein of hMPV and the N protein of RSV show significantly less infiltration of inflammatory cells than that of the infected group without immunization. It can be seen that the group immunized with the recombinant BCG for hMPV P even shows a similar result to that of the uninfected control group (1).

FIG. 4 shows the viral load in lung tissue evaluated by qPCR for the four study groups, expressed as the number of copies of the hMPV N protein per 5,000 copies of β-actin. It is observed that the groups immunized both with the recombinant BCG strain for hMPV P protein and with the recombinant BCG for RSV N have a significantly lower viral load than the unimmunized infected group.

FIG. 5 shows the levels of specific antibodies against RSV (expressed in O.D. 450 nm) evaluated by ELISA in sera of animals immunized with the recombinant BCG-N vaccine, at three different time points: day 0 (pre-immune), day 21 (pre-viral challenge), and day 14 post-infection.

The results show that animals immunized with BCG-N produce anti-RSV antibodies even before the viral challenge. After the challenge with RSV, the levels of specific IgG immunoglobulins against the virus are significantly higher in the group of animals immunized with BCG-N in relation to the control groups.

FIG. 6 shows the isotype characterization of the specific antibodies against RSV induced by the recombinant BCG-N vaccine, expressed in O.D. 450 nm, in sera from immunized and non-immunized animals evaluated by ELISA. The isotype of the immunoglobulins was analyzed by specific secondary antibodies against IgG2a (FIG. 6A) or IgG1 (FIG. 6B) at three different times. FIG. 6C shows the ratio of O.D. 450 nm of IgG2a on IgG1 from data obtained by ELISA at day 14 post-infection. It is shown that the ratio of anti-RSV immunoglobulins of isotype IgG2a/IgG1 after the infection is higher in animals immunized with the vaccine of the invention BCG N.

FIG. 7 shows an RSV-GFP seroneutralization assay in HEp-2 cells. FIG. 7 A HEp-2 cells were treated with a mixture of serum and RSV-GFP previously incubated at 37° C. for 1 hour and after 48 hours they were visualized in an epifluorescence microscope. FIG. 7 B Quantification of plaque-forming units (PFUs) visualized by microscopy. FIG. 7 C Quantification of GFP expression in HEp-2 cells by flow cytometry. The absence of bars in B and C indicates that viral GFP was not detected in the treated cells.

It is evident that the antibodies produced by immunization with the vaccine of the invention are capable of reducing RSV infection in vitro, for which reason these immunoglobulins possess neutralizing functions against the respiratory syncytial virus.

FIG. 8 shows the levels of specific antibodies against hMPV (expressed in O.D. 450 nm), in sera of animals immunized with the recombinant vaccine BCG-N, BCG-P, and unimmunized and uninfected controls (Mock), at two different times: day −21 (pre viral challenge) and day 7 post-infection.

The results show that immunization with recombinant BCG for the RSV N protein induces an increase in the levels of specific IgG against hMPV before and after viral challenge, in the same proportion as immunization with recombinant BCG for P of hMPV.

DETAILED DESCRIPTION OF THE INVENTION

The present invention consists of an immunogenic formulation that can be used for the preparation of vaccines that induce protection against infections caused by respiratory viruses, specifically, respiratory syncytial virus and/or human metapneumovirus, or that attenuate the pathologies caused by these viruses in mammals. The vaccines of the invention contain live attenuated recombinant strains of Mycobacterium, preferably Bacillus Calmette-Guérin (BCG), for example, the BCG Danish or Pasteur strains that recombinantly or heterologously express one or more proteins or immunogenic fragments of RSV. The vaccines of the invention comprise between 1×10⁴-1×10⁹ CFU (colony forming units) of the strains described per dose and can be preserved, before administration, in a lyophilized form or a cold stabilizing saline solution.

Examples of appropriate stabilizer solutions for the immunogenic formulations or vaccines of the invention are:

-   -   Sauton SSI diluted solution (125 μg MgSO₄, 125 μg K₂HPO₄, 1 mg         L-asparagine, 12.5 μg ferric ammonium citrate, 18.4 mg 85%         glycerol, 0.5 mg citric acid, in 1 ml of H₂O) at 4° C.,     -   PBS (137 mM NaCl; 2.7 mM KCl; 4.3 mM Na₂HPO₄; 1.47 mM KH₂PO₄, pH         7.4) supplemented with Tween 80 0.02% and Glycerol 20% at         −80° C. or     -   Volume solution: volume of lactose 25% and Proskauer and Beck's         Medium supplemented with glucose and Tween 80 (PBGT: 0.5 g         asparagine; 5.0 g monopotassium phosphate; 1.5 g magnesium         citrate; 0.5 g potassium sulphate; 0.5 ml Tween 80 and 10.0 g         glucose per liter of distilled water) lyophilized and stored at         a temperature range of between 4° C. and 25° C.

The attenuated recombinant Mycobacterium bacteria of the immunogenic formulation of the present invention contain genes encoding for at least one RSV protein, or immunogenic fragment of the RSV A or RSV B subtypes or both. The respiratory syncytial virus genome has been previously described in the GeneBank database, access numbers NC-001803 and NC-001781.

Where the genes encoding at least one RSV protein, or immunogenic fragment of the RSV A or RSV B subtypes or both of the present invention have at least 80% identity with the genes described in said GeneBank disclosed sequences, access numbers NC-001803 and NC-001781.

The RSV immunogenic proteins, or fragments, corresponding to the RSV NS1, NS2, N, P, M, SH, M2 (ORF1), M2 (ORF2), L, F, or G proteins. In a preferred embodiment, the immunogenic proteins or fragments correspond to N, P, M, SH, M2 (ORF1), M2 (ORF2), L, F or G of RSV.

To obtain the recombinant strains of the invention, the genes that code for these proteins or their immunogenic fragments are inserted into a plasmid, which is incorporated into the bacterium by any available technique. In one embodiment, the plasmid pMV361 is used, it is incorporated into the bacterium by electro transformation, and it is integrated into the bacterial genome by the action of mycobacteriophage integrases (23). These genes can also be inserted into extrachromosomal plasmids, such as pMV261, which is incorporated into Mycobacterium by electrotransformation and remains extrachromosomal in bacteria (23). These genes can be in one or more copies, and their expression is controlled by endogenous, constitutive or inducible, promoters of BCG, for example, the promoter of the hsp60 gene and the promoter of the acr gene respectively. These proteins, or immunogenic fragments of RSV, can be expressed by BCG or other attenuated strains of Mycobacterium, in a soluble-cytoplasmic way, secreted extracellularly or as proteins bound to the cell membrane thanks to the fusion of the genes of the respiratory syncytial virus, or immunogenic fragments, with DNA sequences that code for peptides that function as targeting signals of proteins to the different bacterial compartments, for example, the N-terminal sequence of the gene for the alpha-antigen for extracellular secretion and the N-terminal sequence of the gene for the 19 kD protein for membrane-bound proteins.

The immunogenic formulation disclosed in the present invention can be used in conjunction with immunogenic formulations that contain one or more attenuated strains of Mycobacterium or BCG and that differ in the immunogenic RSV proteins that they express, as well as in the location of the genes (inserted into the genome or extrachromosomal), the number of copies of the protein gene, the promoter that induces the expression of the protein, or the target of the RSV immunogenic protein or fragments (soluble-cytoplasmic, extracellularly secreted or proteins bound to the cellular membrane).

The inventors have shown that the vaccines of the invention induce a Th1-type immune response, which includes both IgG2a isotype antibody-producing B lymphocytes, and an efficient IFN-γ-producing T lymphocyte response. This guarantees humoral protection against these respiratory viruses and an efficient cellular response that enhances both the effectiveness and the applicability of the immunogenic formulation of the invention.

The vaccine of the invention can be administered to the individual subcutaneously, percutaneously, or subdermally, in conjunction with a buffered or physiological saline solution.

As indicated, the immunogenic formulation of the invention can be used to vaccinate individuals who have or have not had previous contact with a respiratory virus such as respiratory syncytial virus or human metapneumovirus, in order to confer protection against these respiratory viruses or to attenuate the pathology caused by these in the future.

The following examples are applicable to immunological formulations containing an attenuated recombinant strain of Mycobacterium expressing the NS2, N, P, M, SH, M2 (ORF1), M2 (ORF2), L, F or G proteins of RSV, and also all combinations of these formulations. Likewise, the examples are applicable to immunological formulations containing one or more attenuated recombinant strains of Mycobacterium; where said recombinant bacteria contain protein genes, or immunogenic fragments of RSV that are inserted in the bacterial genome or in extrachromosomal plasmids, in one or more copies, and their expression is managed by endogenous or exogenous, constitutive or inducible, expressed promoters, in a soluble-cytoplasmic form, secreted extracellularly or as proteins bound to the cell membrane.

EXAMPLES

These examples are illustrative only and are not intended to limit the range of production or application of the invention. Although specific terms are used in the following descriptions, their use is descriptive only and not limiting.

Example I: Immunogenic Formulation Consisting of 10⁸ Bacteria of the Recombinant Danish BCG Strain for the N Gene of RSV Subtype A

The gene is inserted in a copy in the genome of the bacterium under the regulation of the constitutive endogenous promoter hsp60 of BCG and the expression of the protein is cytoplasmic. The immunogenic formulation can be found in a diluted Sauton SSI solution (125 μg MgSO₄, 125 μg K2HPO4, 1 mg L-asparagine, 12.5 μg ferric ammonium citrate, 18.4 mg 85% glycerol, 0.5 mg citric acid in 1 ml of H2O) at −80° C. The formulation can also be found in a PBS solution (137 mM NaCl; 2.7 mM KCl; 4.3 mM Na₂HPO₄; 1.47 mM KH2PO4, pH 7.4), supplemented with 20% Glycerol and 0.02 Tween 80% at a final concentration of 10⁸ bacteria per 100 μl and stored at −80° C. In the same way, the strains can be resuspended in a volume solution: volume of lactose 25% and Proskauer and Beck Medium supplemented with glucose and Tween 80 (PBGT: 0.5 g asparagine; 5.0 g monopotassium phosphate; 1.5 g citrate magnesium; 0.5 g potassium sulfate; 0.5 ml Tween 80 and 10.0 g glucose per liter of distilled water) to later be lyophilized and stored at 25° C.

The Danish BCG strain was transformed by electrotransformation (24) with the plasmid pMV361/N, derived from the plasmid pMV361 (25), which is inserted only once into the genome of the bacterium. This plasmid contains the gene coding for the RSV N protein subtype A, which is expressed under the endogenous and constitutive promoter of the BCG hsp60 gene. The resulting recombinant colonies were grown (at 37° C. in supplemented Middlebrock 7H9 culture medium) until OD_(600nm)=1, they were centrifuged at 4,000 rpm for 20 min (eppendorf rotor model 5702/R A-4-38) and resuspended in solution. PBS (137 mM NaCl; 2.7 mM KCl; 4.3 mM Na₂HPO₄; 1.47 mM KH₂PO₄, pH 7.4), supplemented with 20% Glycerol and 0.02% Tween 80 at a final concentration of 10⁸ bacteria per 100 μl and stored at −80° C. In the same way, the strains can be resuspended in a volume solution: volume of lactose 25% and Proskauer and Beck Medium supplemented with glucose and Tween 80 (PBGT: 0.5 g asparagine; 5.0 g monopotassium phosphate; 1.5 g citrate magnesium; 0.5 g potassium sulfate; 0.5 ml Tween 80 and 10.0 g glucose per liter of distilled water) to later be lyophilized and stored at 25° C. By Western blot, using antibodies to RSV N protein, the inventors observed that this BCG strain recombinantly expresses the RSV N protein subtype A in the cytoplasm. Immunization of BALB/c mice with the described formulation, as a vaccine, confers protection of these animals against an intranasal infection with 10⁷ plaque-forming units of RSV subtype A (FIGS. 1 y 2). This immunogenic formulation can confer immunity against RSV N protein subtype A and B.

Example II: Immunogenic Formulation Consisting of 5×10⁷ Bacteria of the Recombinant Danish BCG Strain for the N Gene of RSV Subtype A and 5×10⁷ Bacteria of the Recombinant Danish BCG Strain for the M2 Gene of RSV Subtype A

In each of the bacteria that make up the immunogenic formulation, the RSV genes are inserted in a copy in the genome of the bacterium under the regulation of the constitutive endogenous promoter hsp60 of BCG and the expression of the protein is cytoplasmic. The immunogenic formulation is preserved in PBS (137 mM NaCl; 2.7 mM KCl; 4.3 mM Na₂HPO₄; 1.47 mM KH₂PO₄, pH 7.4), supplemented with Glycerol 20% and Tween 80 0.02% a final concentration of 10⁸ bacteria per 100 μl and they were stored at −20° C. In the same way, the strains can be resuspended in a volume solution: lactose 25% and Proskauer and Beck Medium supplemented with glucose and Tween 80 (PBGT: 0.5 g asparagine; 5.0 g monopotassium phosphate; 1.5 g citrate magnesium; 0.5 g potassium sulfate; 0.5 ml Tween 80 and 10.0 g glucose per liter of distilled water) to later be lyophilized and stored at 4° C.

The Danish BCG strain was transformed, by electrotransformation (24) with the plasmid pMV361/N or pMV361/M2, derived from the plasmid pMV361 (25), which are inserted only once into the genome of the bacterium. These plasmids contain the genes for the RSV subtype A proteins N and M2 respectively, under the constitutive promoter of the BCG hsp60 gene. The resulting recombinant colonies were grown at 37° C. in supplemented Middlebrock 7H9 culture medium until OD_(600 nm)=1, were centrifuged at 4000 rpm for 20 min (eppendorf rotor model 5702/R A-4-38) and were resuspended in a solution PBS (137 mM NaCl; 2.7 mM KCl; 4.3 mM Na₂HPO₄; 1.47 mM KH₂PO₄, pH 7.4), supplemented with 20% Glycerol and 0.02% Tween 80 at a final concentration of 10⁷ bacteria per 100 μl and stored at −20° C. In the same way, the strains can be resuspended in a volume solution: lactose 25% and Proskauer and Beck Medium supplemented with glucose and Tween 80 (PBGT: 0.5 g asparagine; 5.0 g monopotassium phosphate; 1.5 g citrate magnesium; 0.5 g potassium sulfate; 0.5 ml Tween 80 and 10.0 g glucose per liter of distilled water) to later be lyophilized and stored at 4° C. By Western blot, with the use of antibodies to RSV proteins N and M2, the inventors observed that these BCG Danish strains recombinantly express RSV subtype A proteins N and M2. This immunogenic formulation can confer simultaneous immunity against the M2 and N proteins of RSV subtype A and B.

Example III: Immunogenic Formulation Consisting of 10⁶ Bacteria of the Recombinant Pasteur BCG Strain for a Segment of the F Protein of RSV Subtype B

The gene is found in bacteria extrachromosomally in multiple copies (2-4 copies per bacterium) and codes for a fragment of the RSV subtype B protein F (segment ranging from amino acid 5 to 200). The expression of this gene is under the control of the constitutive endogenous promoter of the gene encoding the alpha-antigen protein (85 kD) of BCG. Furthermore, the protein encoded by this gene has, at its N-terminal end, the peptide signal

HMKKRGLTVAVAGAAILVAGLSGCSSNKSTTGSGETTTTAAGTTASPGG of the 19 kDa protein of BCG, which induces its expression in the bacterial membrane. The immunogenic formulation is preserved in PBS (137 mM NaCl; 2.7 mM KCl; 4.3 mM Na₂HPO₄; 1.47 mM KH₂PO₄, pH 7.4) supplemented with Tween 80 0.02% and Glycerol 20% at −80° C. In the same way, the strains can be resuspended in a volume solution: lactose 25% and Proskauer and Beck Medium supplemented with glucose and Tween 80 (PBGT: 0.5 g asparagine; 5.0 g monopotassium phosphate; 1.5 g citrate magnesium; 0.5 g potassium sulfate; 0.5 ml Tween 80 and 10.0 g glucose per liter of distilled water) to later be lyophilized and stored at 4° C.

The Pasteur BCG strain was transformed, by electrotransformation (24) with the plasmid pMV261/F₅₋₂₀₀, derived from the plasmid pMV261 (25), which resides extrachromosomally in multiple copies in the bacterium. This plasmid encodes a fragment of the F gene of RSV subtype B (a segment that goes from amino acid 5 to 200) fused at its N-terminal end with the peptide signal:

HMKKRGLTVAVAGAAILVAGLSGCSSNKSTTGSGETTTTAAGTTASPGG of the 19 kD protein of BCG, which induces its expression in the bacterial membrane. The expression of this gene is under the control of the constitutive endogenous promoter of the gene encoding the alpha antigen protein (85 kD) of BCG. The resulting recombinant colonies were grown up to OD_(600 nm)=1, at 37° C. in supplemented Middlebrock 7H9 culture medium and were centrifuged at 4,000 rpm for 20 min (eppendorf rotor model 5702/R A-4-38) and resuspended in PBS (137 mM NaCl; 2.7 mM KCl; 4.3 mM Na₂HPO₄; 1.47 mM KH₂PO₄, pH 7.4) supplemented with Tween 80 0.02% and Glycerol 20% at a final concentration of 10⁶ bacteria per 100 μl. In the same way, the strains can be resuspended in a volume solution: volume of lactose 25% and Proskauer and Beck Medium supplemented with glucose and Tween 80 (PBGT: 0.5 g asparagine; 5.0 g monopotassium phosphate; 1.5 g citrate magnesium; 0.5 g potassium sulfate; 0.5 ml Tween 80 and 10.0 g glucose per liter of distilled water) to later be lyophilized in aliquots with 10⁶ bacteria each and stored at 4° C. This immunogenic formulation can confer immunity against the F protein of RSV subtype A and B.

Example IV: Immunogenic Formulation Consisting of 10⁵ Bacteria of the Recombinant Danish BCG Strain Simultaneously for the N and M2 Genes of RSV Subtype A

The N gene is inserted in a copy in the genome of the bacterium under the regulation of the constitutive endogenous promoter hsp60 of BCG and the expression of the protein is cytoplasmic. The M2 gene is found extrachromosomally in the bacterium in multiple copies (2-4 copies per bacterium) under the control of the constitutive endogenous promoter of the gene encoding the alpha antigen protein (85 kD) of BCG. The protein encoded by the M2 gene has, at its N-terminal end, the peptide signal

HMKKRGLTVAVAGAAILVAGLSGCSSNKSTTGSGETTTTAAGTTASPGG of the 19 kDa protein of BCG, which induces its expression in the bacterial membrane. The immunogenic formulation is preserved at 4° C. lyophilized from the bacteria resuspended in a solution volume: lactose 25% and Proskauer and Beck Medium supplemented with glucose and Tween 80 (PBGT: 0.5 g asparagine; 5.0 g monopotassium phosphate; 1.5 g magnesium citrate; 0.5 g potassium sulfate; 0.5 ml Tween 80 and 10.0 g glucose per liter of lyophilized distilled water stored at 4° C. Similarly, the strains can be preserved in a PBS solution (137 mM NaCl; 2.7 mM KCl; 4.3 mM Na₂HPO₄; 1.47 mM KH₂PO₄, pH 7.4) supplemented with Tween 80 0.02% and Glycerol 20% at a final concentration of 10′ bacteria per 100 μl.

The Danish BCG strain was transformed by electrotransformation (24) with the plasmid pMV361/N, derived from the plasmid pMV361 (25), which is inserted only once into the genome of the bacterium. This plasmid contains the gene coding for the RSV N protein subtype A, which is expressed under the endogenous and constitutive promoter of the BCG hsp60 gene. After verifying that the resulting BCG strain is recombinant for the RSV N protein, it was transformed by electrotransformation (24) with the plasmid pMV206/M2, derived from the plasmid pMV206 (25), which resides extrachromosomally in multiple copies in the bacteria. The protein encoded by the M2 gene has, at its N-terminal end, the peptide signal

HMKKRGLTVAVAGAAILVAGLSGCSSNKSTTGSGETTTTAAGTTASPGG of the 19 kD protein of BCG, which induces its expression in the bacterial membrane. The resulting recombinant colonies were grown (at 37° C. in supplemented Middlebrock 7H9 culture medium) until OD_(600 nm)=1, were centrifuged at 4,000 rpm for 20 min (eppendorf rotor model 5702/R A-4-38) and were resuspended in a solution volume: lactose 25% and Proskauer and Beck's Medium supplemented with glucose and Tween 80 (PBGT: 0.5 g asparagine; 5.0 g monopotassium phosphate; 1.5 g magnesium citrate; 0.5 g sulfate of potassium; 0.5 ml Tween 80 and 10.0 g glucose per liter of distilled water) at a final concentration of 10′ bacteria per 1 ml. Finally, 1 ml aliquots were lyophilized with 10⁵ bacteria and the aliquots were stored at 4° C. Similarly, the strains can be preserved in a PBS solution (137 mM NaCl; 2.7 mM KCl; 4.3 mM Na₂HPO₄; 1.47 mM KH₂PO₄, pH 7.4) supplemented with Tween 80 0.02% and Glycerol 20% at a final concentration of 10⁵ bacteria per 100 μl. This immunogenic formulation can confer immunity against the N and M2 proteins of RSV subtype A and B.

Example V: Immunogenic Formulation Consisting of 10⁴ Bacteria of the Recombinant Danish BCG Strain for the N Gene of RSV Subtype A

The gene is inserted in one copy in the bacterial genome under the regulation of the endogenous inducible acr promoter of BCG, which is active in response to nitric oxide, low oxygen concentrations, and stationary phases of growth. The expression of the protein is cytoplasmic. The immunogenic formulation is lyophilized from a volume: volume solution of 25% lactose and Proskauer and Beck Medium supplemented with glucose and Tween 80 (PBGT: 0.5 g asparagine; 5.0 g monopotassium phosphate; 1.5 g citrate. magnesium; 0.5 g potassium sulfate; 0.5 ml Tween 80 and 10.0 g glucose per liter of distilled water) stored at 25° C. in dilute Sauton SSI solution (125 μg MgSO₄, 125 μg K₂HPO₄, 1 mg L-asparagine, 12.5 μg ferric ammonium citrate, 18.4 mg 85% glycerol, 0.5 mg citric acid in 1 ml of H₂O). Similarly, the strains can be preserved in a PBS solution (137 mM NaCl; 2.7 mM KCl; 4.3 mM Na₂HPO₄; 1.47 mM KH₂PO₄, pH 7.4) supplemented with Tween 80 0.02% and Glycerol 20% at a final concentration of 10⁴ bacteria per 100 μl.

The Danish BCG strain was transformed by electrotransformation (24) with the plasmid pMV361_(Pacr)/N, derived from the plasmid pMV361 (25), which is inserted only once into the genome of the bacterium. This plasmid contains the gene that codes for the RSV N protein subtype A, which is expressed under the endogenous and inducible promoter of the BCG acr gene (26). The resulting recombinant colonies were grown (at 37° C. in supplemented Middlebrock 7H9 culture medium) until OD_(600 nm)=1, were centrifuged at 4,000 rpm for 20 min (eppendorf rotor model 5702/R A-4-38) and were resuspended in a solution volume: lactose 25% and Proskauer and Beck's Medium supplemented with glucose and Tween 80 (PBGT: 0.5 g asparagine; 5.0 g monopotassium phosphate; 1.5 g magnesium citrate; 0.5 g sulfate of potassium; 0.5 ml Tween 80 and 10.0 g glucose per liter of distilled water). Finally, 1 ml aliquots were lyophilized with 10⁴ bacteria and stored at 25° C. Similarly, the strains can be preserved in a PBS solution (137 mM NaCl; 2.7 mM KCl; 4.3 mM Na₂HPO₄; 1.47 mM KH₂PO₄, pH 7.4) supplemented with Tween 80 0.02% and Glycerol 20% at a final concentration of 10⁸ bacteria per 100 μl. This immunogenic formulation can confer immunity against RSV N protein subtype A and B.

Example VI: Immunogenic Formulation Consisting of 10⁹ Bacteria of the Recombinant Danish BCG Strain for the N Gene of RSV Subtype A

The gene is inserted in a copy in the genome of the bacterium under the regulation of the exogenous promoter of phage T7 of constitutive expression in strains of BCG that co-express the polymerase of phage T7. The expression of the protein is cytoplasmic. The immunogenic formulation is in a diluted Sauton SSI solution (125 μg MgSO₄, 125 μg K₂HPO₄, 1 mg L-asparagine, 12.5 μg ferric ammonium citrate, 18.4 mg 85% glycerol, 0.5 mg citric acid in 1 ml of H₂O) and was stored at −20° C., or it can be lyophilized and stored at 4° C. Similarly, the strains can be preserved in a PBS solution (137 mM NaCl; 2.7 mM KCl; 4.3 mM Na₂HPO₄; 1.47 mM KH₂PO₄, pH 7.4) supplemented with Tween 80 0.02% and Glycerol 20% at a final concentration of 10⁹ bacteria per 100 μl.

The Danish BCG strain was transformed by electrotransformation (24) with the plasmid pMV361_(PT7)/N, derived from the plasmid pMV361 (25), which is inserted only once into the genome of the bacterium. This plasmid contains the gene encoding the RSV N protein subtype A, which is expressed under the T7 promoter activated by the expression of phage T7 polymerase (27).

The resulting BCG strain was transformed by electrotransformation (24) with the plasmid pMV261_(Amp)/PolT7, derived from the plasmid pMV261 (25), which resides extrachromosomally in the bacterium in multiple copies. In this plasmid, resistance to the antibiotic kanamycin (27) has been replaced by resistance to the antibiotic hygromycin (Higr). The T7 polymerase of phage T7 is under the control of the constitutive promoter of the BCG hsp60 gene. The resulting recombinant colonies were grown at 37° C. in supplemented Middlebrock 7H9 culture medium until OD_(600 nm)=1, centrifuged at 4000 rpm for 20 min (eppendorf rotor model 5702/R A-4-38) and resuspended in a solution diluted Sauton SSI (125 μg MgSO₄, 125 μg K₂HPO₄, 1 mg L-asparagine, 12.5 μg ferric ammonium citrate, 18.4 mg 85% glycerol, 0.5 mg citric acid in 1 ml of H₂O) and was stored at −80° C. This immunogenic formulation can confer immunity against RSV N protein subtype A and B.

Example VII: Protection from RSV BCG-N, Against hMPV Infections

To determine that the vaccines of the invention provide protection against human metapneumovirus (hMPV), mice previously immunized with one of the vaccines of the invention or with a recombinant BCG vaccine for the P protein of hMPV effective against infection by hMPV. These results were compared with mice infected with hMPV without prior immunization and with mice without infection.

The experimental groups, of 3 mice each, are the following:

-   -   1) Control Uninfected, unimmunized     -   2) Infected with hMPV without immunization     -   3) Immunized with hPMV BCG-P, infected with hMPV     -   4) Immunized with RSV BCG-N, infected with hMPV

First, the infiltrate of polymorphonuclear cells in the lung and bronchoalveolar lavage (BAL) was analyzed by flow cytometry for the 4 groups, determining the percentage of CD11c⁻/CD11b⁺/Gr1⁺ polymorphonuclear cells. The presence of these cells is directly correlated with the inflammatory response to viral infection. FIG. 3 -A shows the percentage of CD11c⁻/CD11b⁺/Gr1⁺ cells in BAL for the four groups studied. It is observed that the groups immunized with BCG-P and BCG-N show significantly less inflammatory reaction than the infected group without immunization.

FIG. 3 -B shows the percentage of CD11c⁺/CD11b⁺/Gr1⁺ cells in the lung for the four groups studied. As in the previous case, it is observed that the groups immunized with BCG-P and BCG-N show infiltration of inflammatory cells significantly less than that of the infected group without immunization. It can be seen that group 3, immunized with BCG-P, shows a similar result to that of the uninfected group (1).

Second, the viral load in lung tissue of the 4 groups was determined by quantitative PCR (qPCR) (FIG. 4 ), expressed as the number of copies of the hMPV N protein per 5,000 copies of β-actin. It is observed that the immunized groups have a significantly lower viral load than the unimmunized infected group.

As can be appreciated, the vaccine of the invention provides similar protection against an hMPV infection, to the protection granted by a recombinant BCG vaccine for a protein of the same hMPV virus, in this case, the P protein.

Example VIII: Induction of Humoral Response by BCG RSV-N Vaccine

To determine that the vaccines of the invention induce a Th1-type response, BALB/cJ mice aged 6 to 8 weeks received a subcutaneous injection in the back with 1×10⁸ CFU of BCG WT or recombinant BCG that expresses the RSV-N protein, in a final volume 100 μL per dose. After 14 days, they were given a booster with the same starting dose. After 21 days from the first injection of BCG WT or BCG-N, the animals were challenged, for which RSV A2 strain 13018-8 obtained from a clinical isolate was used. Pre-immune serum samples were obtained (day 0), before viral challenge (day 21), and 14 days after infection. These samples were analyzed by ELISA at a 1/500 dilution. The results show that animals immunized with BCG-N produce anti-RSV antibodies even in stages prior to viral challenge, this production is significantly higher than unimmunized controls and animals immunized with BCG WT. After the challenge with RSV, the levels of specific IgG immunoglobulins against the virus show an even greater increase that is maintained at day 14 post-infection (FIG. 5 ).

To establish the isotype of the immunoglobulins produced by immunization, the levels of RSV-specific IgG1 and IgG2a isotype antibodies were analyzed by ELISA in the serum of the test animals. From day 7 post-infection, a significantly greater increase in the production of IgG2a isotype antibodies is observed in animals immunized with the BCG RSV-N vaccine of the invention, compared to control animals, which includes animals vaccinated with wild-type BCG, and not immunized. On the other hand, IgG1 isotype anti-RSV immunoglobulins show an increase in animals vaccinated with BCG-N prior to infection, however, the RSV control group and animals vaccinated with BCG WT already manage to match their production towards day 14 after viral challenge (FIG. 6 -A, 6-B). The latter is better evidenced in FIG. 6 -C, where it is shown that the ratio of anti-RSV immunoglobulins of isotype IgG2a/IgG1 is higher in animals immunized with the BCG RSV-N vaccine of the invention than in the control group only infected with RSV. As we have already pointed out, this relationship is indicative of an immune response polarized towards a Th1 phenotype.

In order to determine if the humoral response induced by vaccination was capable of protecting against RSV infection, after inactivating complement, the RSV-GFP virus was incubated with sera from animals from all experimental groups on day 14 post-infection and prior to viral challenge, and then HEp-2 cells were infected with these mixtures. In FIG. 7 , in the epifluorescence microscopy images, a decrease in the expression of RSV-GFP in the cells previously treated with serum from animals immunized with BCG-N in relation to the other treatments is observed. When quantifying plaque-forming units, a significant decrease in this parameter is evidenced, only in the treatment with serum from day 14 post-infection of animals immunized with BCG-N (FIG. 7 -B) and when analyzing the expression of RSV-GFP by flow cytometry, the same trend is evidenced (compare FIG. 7 -C with 7-B).

Example IX: Production of Anti-hMPV IgG Antibodies in Animals Immunized with RSV BCG-N

In order to evaluate the production of specific immunoglobulins against hMPV, serum samples were obtained from BALB/cJ animals challenged with this virus and previously immunized with the vaccine of the invention, recombinant BCG for the RSV N protein (BCG-N) or with a recombinant BCG vaccine for the hMPV P protein (BCG-P). These results were compared to animals infected with hMPV without prior immunization and with an uninfected control (Mock). The serum are compared at 2 different times: previral challenge and day 7 post-infection. The samples were analyzed by ELISA at a 1/1,000 dilution. The results are seen in FIG. 8 , where it is shown that the total anti-hMPV IgG immunoglobulins significantly increased in the vaccinated animals compared to the control groups.

It is highly significant that animals immunized with both RSV BCG-N and hMPV BCG-P achieve the same levels of specific antibodies against human metapneumovirus, before and after viral challenge. This means that the vaccines of the invention, which include RSV proteins, provide protection against human metapneumovirus, similar to that obtained by a specific vaccine for that virus based on a recombinant BCG for the P protein of hMPV.

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1-11. (canceled)
 12. Use of an immunogenic formulation containing the Bacillus strain of Calmette and Guerin (BCG), at a concentration between 10⁴-10⁹ bacteria, expressing at least one protein or immunogenic fragment of respiratory syncytial virus (RSV), in a pharmaceutically acceptable saline buffer solution wherein the RSV immunogenic protein or fragment corresponds to the RSV nucleocapsid protein (N protein) and serves to prepare a vaccine useful for preventing, treating, or attenuating human metapneumovirus infections.
 13. Use according to claim 12 where the RSV immunogenic protein or fragment corresponds to the N protein of RSV A or RSV B subtypes or both subtypes wherein it serves to prepare a vaccine useful for preventing, treating, or attenuating human metapneumovirus infections.
 14. Use according to claim 13 where the genes encoding the proteins or immunogenic fragments of the RSV N protein are inserted into the genome of Mycobacterium, preferably BCG or in extrachromosomal plasmids, in one or more copies wherein it serves to prepare a vaccine useful for preventing, treating, or attenuating human metapneumovirus infections.
 15. Use according to claim 14 where the expression of protein genes are commanded by endogenous or exogenous promoters of Mycobacterium BCG, constitutive or inducible wherein it serves to prepare a vaccine useful for preventing, treating, or attenuating human metapneumovirus infections.
 16. Use according to claim 12 where the immunogenic RSV proteins or fragments corresponding to the RSV N protein can be expressed by BCG in a soluble-cytoplasmic form, secreted extracellularly or as cell membrane-bound proteins wherein it serves to prepare a vaccine useful for preventing, treating, or attenuating human metapneumovirus infections.
 17. Use according to claim 12 where the formulation can be used in conjunction with other immunogenic formulations, expressing proteins or immunogenic fragments different from RSV N wherein it serves to prepare a vaccine useful for preventing, treating, or attenuating human metapneumovirus infections.
 18. Use according to claim 12 where the formulation can be used in conjunction with other immunogenic formulations, which differ in the form expressed by immunogenic proteins or fragments, soluble, soluble-cytoplasmic, extracellularly secreted or proteins bound to cell membrane wherein it serves to prepare a vaccine useful for preventing, treating, or attenuating human metapneumovirus infections.
 19. Use according to claim 12 where the formulation can be used in conjunction with other immunogenic formulations or differing in the number of copies of RSV protein genes or immunogenic fragments, as well as in the way in which the expression of RSV proteins or immunogenic fragments is controlled, constitutive or inducible and also differing in the destination of the RSV protein or immunogenic fragment wherein it serves to prepare a vaccine useful for preventing, treating, or attenuating human metapneumovirus infections.
 20. Use according to claim 12 where the formulation can be stabilized by freezing, freeze-drying or saline buffer for preservation prior to use wherein it serves to prepare a vaccine useful for preventing, treating, or attenuating human methamneumovirus infections.
 21. Use according to claim 12 where the formulation can be administered subcutaneously, percutaneously or subdermally in acceptable physiological saline solution wherein it serves to prepare a vaccine useful for preventing, treating, or attenuating human metapneumovirus infections. 